Evaluating the Utility of Canine Mdr1 Knockout Madin-Darby Canine Kidney I Cells in Permeability Screening and Efflux Substrate Determination

Permeability
assays are commonly conducted with Madin-Darby canine
kidney (MDCK) cells to predict the intestinal absorption of small-molecule
drug candidates. In addition, MDCK cells transfected to overexpress
efflux transporters are often used to identify substrates. However,
MDCK cells exhibit endogenous efflux activity for a significant proportion
of experimental compounds, potentially leading to the underestimation
of permeability and confounded findings in transport studies. The
goal of this study was to evaluate canine Mdr1 knockout MDCK (gMDCKI)
cells in permeability screening and human MDR1 substrate determination
in a drug discovery setting. The gMDCKI cells were established by
CRISPR-Cas9-mediated knockout of the canine Mdr1 gene in MDCKI wildtype
(wt) cells. A comparison of efflux ratios (ER) between MDCKI wt and
gMDCKI showed that out of 135 compounds tested, 38% showed efflux
activity in MDCKI wt, while no significant efflux was observed in
gMDCKI cells. Apparent permeability (Papp) from apical-to-basolateral
(A-to-B) and basolateral-to-apical were near unity in gMDCKI cells,
which approximated passive permeability, and 17% of compounds demonstrated
increases in their Papp A-to-B values. Overexpression of human MDR1
in gMDCKI (gMDCKI-MDR1) cells enabled substrate determination without
the contribution of endogenous efflux, and the assay was able to deconvolute
ambiguous results from MDCKI-MDR1 and identify species differences
in substrate specificity. An analysis of 395 and 474 compounds in
gMDCKI and gMDCKI-MDR1, respectively, suggested physicochemical properties
that were associated with low permeability correlated with MDR1 recognition.
Poorly permeable compounds and MDR1 substrates were more likely to
be large, flexible, and more capable of forming external hydrogen
bonds. On the basis of our evaluation, we concluded that gMDCKI is
a better cell line for permeability screening and efflux substrate
determination than the MDCK wt cell line.